Project Summary/Abstract Feeding is an important biological process essential for animal survival. Neuropeptides are heavily implicated in signaling pathways related to feeding, but their specific functions remain elusive. This lack of understanding is partly due to their activity being heavily co-modulated by other neuropeptides, necessitating a global analysis that can study all neuropeptides in a sample at the same time. Other major challenges with neuropeptide research stem from difficulties with detection due to their low abundance in complex biological matrices. This research strives to bridge these knowledge gaps in order to study the changes in the entire neuropeptidome in response to feeding. The crustacean species Cancer borealis will be used as a model organism due to its simplified and electrophysiologically well-characterized nervous system and the homology of its neuropeptides to those of higher order organisms. Feeding-related processes are co-modulated by multiple neuropeptides whose functions are dependent upon one another, and thus, studies investigating only a small subset of these neuropeptides (e.g. using antibodies) are insufficient. A multifaceted mass spectrometry (MS) method will be employed in order to profile the neuropeptides present in the circulating fluid (hemolymph) and neurosecretory tissue to determine changes due to feeding. It is hypothesized that neuropeptides implicated in the feeding process will experience a change in abundance at some time point during the feeding process, while neuropeptides unrelated to feeding will remain unchanged. To test this hypothesis, hemolymph will be sampled from animals at several time points over the course of feeding, and neuropeptides will be quantified to determine changes in abundance (Aim 1). Hemolymph will be sampled directly using a needle and a syringe, as well as with in vivo microdialysis to continuously monitor secreted neuropeptide changes over the course of time. Neuropeptides will also be quantified in neurosecretory tissue (e.g. pericardial organs (POs) and sinus glands) at several time points over the course of feeding (Aim 2). POs will also be imaged using MS imaging in order to determine changes in localization as a result of feeding. Changes in either abundance or localization will be correlated with changes in abundance observed in hemolymph to better understand neuropeptide secretion. A method will also be developed to gain a deeper profiling of the neuropeptidome by incorporating capillary electrophoresis (CE) with matrix-assisted laser desorption/ionization mass spectrometry and ion mobility in order to achieve superior separation and sensitivity for detecting low-abundance neuropeptides (Aim 3). The method is expected to provide complementary detection capabilities to conventional methods (i.e. liquid chromatography-electrospray ionization-MS), thus enabling identification of previously unidentified neuropeptides. The combined in-depth profiling of feeding-related neuropeptides will allow for specific functional studies to be performed via electrophysiology, thus providing knowledge to advance the overall field of neuropeptide research and facilitate the development of pharmaceuticals to treat feeding-related disorders.